Method and equipment to monitor nucleic acid hybridization on a DNA chip using four-dimensional parameters

ABSTRACT

This invention is to provide a method for detecting DNA hybridization on DNA chips using 4-D parameters. Based on the traditional method of using 3-D parameters to detect DNA hybridization on DNA chips, this invention introduces temperature as the forth parameters to determine the melting temperatures by scanning the changes of fluorescence intensity of the fluorescent labels on the double-stranded oligo nuclei acids resulted from the increasing of hybridization temperature. Comparing the measured melting temperatures with the standard melting temperature specific for each probe on the chip is used to gain insight into the specific property of the single-stranded nucleic acids in the sample. This device is composed of a DNA chip containing transparent glass chamber, in which a temperature sensor and a thermo-circler are installed. This invention provides a specific, sensitive, and easy operating method to detect gene in a complex sample. The device used in this invention has simple structure, and can be coupled with various types of DNA chips with low cost.

FIELD OF THE INVENTION

This invention is about gene detection technologies emphasized on thespecific methods and equipments to monitor nucleic acid hybridization ona DNA chip using four-dimensionalparameters.

BACKGROUND OF THE INVENTION

In 1953, Watson and Crick suggested the concept of double stranded DNA.They had some significant discoveries. (1) DNA molecules were composedof two anti-parallel poly-nucleic acid chains. (2) There were rules forparing the four bases—chargaff et al. analyzed the base compositions ofDNA molecules by chromatograph from many organisms, and found that thenumbers of A and T were equal, while the numbers of C and G were alsoequal. So they suggest there exist four possible base pairs: A-T, T-A,G-C and C-G. (3) The connection of the two chains were through hydrogenbounds—the surface of the base pairs goes through and was roughlyperpendicular to the axis. Two and three hydrogen bounds can formbetween the A-T and G-C pairs, respectively. Meanwhile, hydrophobicforce also contributes to stabilize the DNA double helixes. (4) Becauseall the base pairs follow these rules, every chain can have randomsequences. However, once the sequence of one of the chains isdetermined, the other one must have the corresponding nucleotidesequences.

As the DNA double helix is maintained by both hydrogen bound andhydrophobic force, factors, such as heat, pH, organic solvent, etc.,which can destroy hydrogen and hydrophobic bounds, would denature DNAdouble helixes to random single chain threads. The annealing betweendenatured DNA single chains through pairing is called hybridization.Hybridization can occur between homologous DNA molecules as well ashomologous DNA and RNA molecules. During hybridization, the twocomplementary single-stranded DNA chains form double-stranded hybridsthrough non-covalent bounds. When the sequence of one of the chains isknown, the existence of its complementary chain in an unknown DNA samplecan be detected through hybridization.

Based on the above principle, many gene products have been developed,among which gene sensor has many applications.

Recently, the research in the field of DNA sensor (DNA or nucleic acidsensor) has become the hot spot of research. Gene sensor, as a simple,fast, and cheap detection method, has many potential applications in thefields of molecular biology, medical analysis, and environmentmonitoring. It can also be applied to study DNA-drug and protein-proteininteractions in addition to sequence analysis, mutation detection, genedetection, and clinical diagnostics.

The method for gene analysis to analyze DNA sequences in non-homogenoussystem is now through DNA hybridization. Hybridization can occur betweenhomologous DNA molecules as well as homologous DNA and RNA molecules.During hybridization, the two complementary single-stranded DNA chainsform double-stranded hybrids through non-covalent bounds. When thesequence of one of the chains is known, the existence of itscomplementary chain in an unknown DNA sample can be detected throughhybridization. The most common method is to fix a single DNA chain withknown sequence information on a solid surface, and use it to hybridizeto the complementary single chains in the sample buffer to detect theexistence of the wanted DNA molecule in the liquid phase.

Recently, such research is getting deeper and deeper, and there is greatvalue in DNA detection through hybridization. The major applicationsrely in the fields of clinical diagnostics, forensic science, foodindustry, biochemistry, environment protection, etc. The application ofusing non-radioactive labels such as biotin, digosin, and fluorescentdyes has made the detection more convenient and safer. In particular,the application of PCR amplification has made the method very sensitive.

The traditional DNA hybridization reaction requires a labeling step todetect hybridization signals, which allow in situ detection, and canachieve high sensitivity. For example, PCR technology can reach thedetection limit at the nmol/l range. Bioinformatics also provides meansto detect a specific DNA sequence from a complex mixture of DNA. Becauseof using short wave fluorescence and co-focal microscopy, fluorescentlabeling has become the routine method for detecting nano-amount of DNAmolecules. Among the available fluorescent labeling methods, the deviceof DNA chip (gene chip or DNA microarray) only include the XYZthree-dimensional (3-D) parameters, such as the Affymetrix GeneChip. Onthese chips oligo nucleic probes are fix on glass surface, and theirbase composition and chain length can be represented by XYZ 3-Dparameters. The single-stranded oligo target sequences are directlylabeled with fluorescent dyes, hybridized to the probes on the chip toform double helixes, followed by the detection by using a scanner toobtain sample information. The U.S. Pat. No. 5,445,934 and U.S. Pat. No.5,744,305, disclosed the technologies and device of usingphotolithography to fabricate high-density DNA probes on a DNA chip. Insuch device the hybridization between all the fixed probes on the chipsand the other DNA molecules in the sample buffer was carried out at thesame temperature. Because of the differences between the probe lengths,base composition (GC content), the Tm (melting temperature), whichrepresents the temperature when 50% of the probes and their targets areseparated, are different. Therefore, the optimum hybridizationtemperatures are different for each probe. Because of such discrepancyin hybridization temperature, the accuracy of results cannot beguaranteed, and single base mismatch cannot be detected. To overcomesuch disadvantages, U.S. Pat. No. 6,238,868 described a type ofmicroarrays by introducing electric filed as a free parameter toexpedite the hybridization procedure. However, this technology iscomplicated, high cost, and requires the labeling of oligo targetsequences or hybridizing with another reporter probes with fluorescentdyes. As a result it cannot guarantee the accuracy of results, andlimited the application of hybridization microarrays.

Recently, some researchers begin to use non-labeling methods to analyzegene sequences. The most popular one is the DNA biosensor system. Thesebiosensors can be categorized into three classes: (1) optical biosensor,which can be further divided into three classes of fluorescent opticalfiber gene sensor, surface enhance Raman gene probe and surface plasmonresonance gene sensor, (2) electro-chemistry biosensor, and (3) piezogene biosensor. Further information of their specificity and sensitivityis still waiting.

DETAILED DESCRIPTION OF THE INVENTION

The purpose of this invention is to provide a method for detecting DNAhybridization on DNA chips using 4-D parameters. Based on thetraditional method of using 3-D parameters to detect DNA hybridizationon DNA chips, this invention introduces temperature as the fourthparameters to determine the melting temperatures by scanning the changesof fluorescence intensity of the fluorescent labels on thedouble-stranded oligo nuclei acid resulted from the increasing ofhybridization temperature. This method is simple, accurate, sensitiveand specific.

Another purpose of this invention is to provide a device for detectingDNA hybridization on DNA chips using 4-D parameters. This device issimple, low cost, and can guarantee the accuracy of results fromhigh-density microarrays even with the discrepancy in hybridizationtemperature.

This invention is achieved through the following methods:

Method to monitor nucleic acid hybridization on a DNA chip usingfour-dimensional parameters includes the following steps:

(1) Place the DNA chip in a temperature-controlled chamber;

(2) Incubate the chip with the single-stranded target oligos and thedouble-stranded chains inserted with the fluorescent dye in a reactionbuffer, lower the temperature to the annealing temperature (Th) to allowfor the hybridization between the probes on the chip and thesingle-stranded targets, which results the labeling of the doublehelixes with the fluorescent dye;

(3) Increase the temperature in the device from Th to 100° C. at thespeed of 0.001–1° C./sec, during which at each ΔT° C. (defined as thetemperature difference per second of increasing) the chip was scanned bya scanner to obtain the fluorescent intensity F; When the temperature inthe device reaches the melting temperature Tm, the double strandsdisassociate, the fluorescent dye diffuses into the buffer, resulting ina rapid loss of fluorescent intensity; The scanning of fluorescentintensity of every probe on the chip records the turning point on acontinuous melting curve of the double-stranded DNA, or on a derivativecurve, which must provide the Tm of the double-stranded molecules at thepeak position on the curves; By comparing the above Tm with thecalculated Tm of known sequences, the property of the oligo single chainthat hybridizes with the surface probes can be obtained.

In preferred embodiments, to wash off the excess target oligo chains andreaction buffer, a washing step at annealing temperature with bufferfree of fluorescent dyes was also included in the above item (2).

The preferred conditions are as follows:

-   -   Annealing temperature Th is at 4–89° C.;    -   Melting temperature Tm is at 8–100° C.;    -   Speed of temperature increasing is 0.01–1° C./sec.

The above reaction buffer of fluorescent dye is one of the SYBR Green I,SYBR Green II, and SYBR Gold buffers (Molecular Probe, USA).

Another purpose of this invention can be achieved using the followingsteps:

Device to monitor nucleic acid hybridization on a DNA chip usingfour-dimensional parameters includes the container for DNA chips, DNAchips, temperature controlled thermocycler, and the buffer input andoutput; The DNA chips are placed in the chip container, which isconnected to the thermocycler.

The above temperature controlled thermocycler is composed of temperaturesensor, thermocycler and temperature controlling devices; of these, thetemperature sensor is connected to the temperature controlling devicewhich is also connected to the thermocycler.

This invention further provides practical applications of using thedevice for monitoring nucleic acid hybridization on a DNA chip usingfour-dimensional parameters.

The device to monitor nucleic acid hybridization on a DNA chip usingfour-dimensional parameters can also be used to analyze and separate DNAsamples.

The device to monitor nucleic acid hybridization on a DNA chip usingfour-dimensional parameters can also be used to analyze and separate RNAsamples.

This invention has the following advantages over the existingtechnologies:

1. Based on the traditional method of using 3-D parameters to detect DNAhybridization on DNA chips, this invention introduces temperature as thefourth parameters to determine the melting temperatures by scanning thechanges of fluorescence intensity of the fluorescent labels on thedouble-stranded oligo nuclei acids resulted from the increasing ofhybridization temperature. This method is simple, accurate, sensitiveand specific.

2. This invention introduces the temperature scanning method to solvethe problem whether the results are accurate caused by the discrepancyin hybridization temperature, and provides simple and definitive yes/noanswers to the results.

3. The target oligo sequences used in this invention does not require alabeling step nor another fluorescence-labeled reporter probe, whichsimplified the operation.

4. The device in this invention has simple structure, and can be coupledwith various commercially available chips to reduce the cost; Even withdifferences in hybridization temperatures, this invention stillguarantees the accuracy of results from high-density microarray assays.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. The structure of the device of this invention.

FIG. 2A. The melting temperature curves (F˜T) obtained by scanning theDNA chips using the device of this invention.

FIG. 2B. The derivative melting temperature curves (F˜T) obtained byscanning the DNA chips using the device of this invention.

DESCRIPTION OF PREFERRED EMBODIMENT

FIG. 1 describes the device to monitor nucleic acid hybridization on aDNA chip using four-dimensional parameters. As shown in the figure,Device 9 includes a transparent glass box 1, a commercially availableDNA chip 2, a Temperature Controlled Thermocycler Device 3, a bufferinput 5 and buffer output 7. The Temperature Controlled ThermocyclerDevice 3 includes a Temperature Sensor 4, a Thermocycler 8 and aTemperature Controlling Device 6. The Temperature Controlling Device 6is a temperature controlling computer. Device 4 and Device 6 areconnected, and Device 6 and Device 8 are also connected. The temperaturecontrolling computer 6 is programmed to control the temperature of thebuffer and DNA chips 2 in the glass chamber 1 through the TemperatureSensor 4 and the Thermocycler Device 8.

Based on this invention, the single-stranded target oligos and thedouble-stranded chains inserted with the fluorescent dye are mixed in ahybridization buffer and added to the glass reaction chamber 1. Thetemperature in chamber 1 is rapidly lowered to the annealing temperature(Th=4˜89° C.), and chamber is washed with phosphate buffer to remove theexcess oligos and reaction buffer. Use thermocycler 8 to increase thetemperature in chamber 1 from Th to 100° C. at the speed of 0.01°C./sec, during which at each 0.01° C. increase the chip 2 was scanned bya scanner to obtain the fluorescent intensity F. As long as the Th islow enough, single-stranded oligos in the sample will form the doublehelixes with the probes on the chip, resulting in the insertion of highconcentration of SYBR Green I dye into the double-stranded oligos.Excited by laser at 470–490 nm, the dye will emit specific fluorescenceat the wave length of 530 nm. With the temperature increasing to themelting temperature Tm (Tm=8–100° C.), double-stranded oligos melt torelease the single-chained target oligos from the corresponding probeson the chip 2, which leads to the diffusion of SYBR Green I into thebuffer that causes the rapid loss of fluorescent signals. The turningpoints on the melting temperature curves (F˜T) (FIG. 2A) or thederivative melting curve (dF/dT˜T)(FIG. 2B) obtained by scanning the DNAchips will define the melting temperature Tm. In FIG. 2A, F representsthe fluorescent intensity of the probe, T represents temperature, Curve1 is the melting curve of a double helix with mismatch, and Curve 2represent the melting curve of a perfect match. In FIG. 2B, dF/dT is thederivative of fluorescent intensity to temperature, where T representsthe temperature; Curve 3 is the melting curve of a double helix withmismatches; Tm represents the temperature at the peak of the curve,namely the melting temperature; Curve 4 represent the melting curve of aperfect match. Tmp, namely the melting temperature, which is at the peakposition of the curve, also represents the specific melting temperatureof the probe. Tmp can be obtained using the standard samples withperfect matches to the probe. When Tm is less than Tmp, we can concludethat there exist mismatches between the target oligos and the probe,because their binding force is less than that with perfect matches. Onlywhen Tm is equal to Tmp can one tell that the pairing is perfect.

INDUSTRIAL APPLICATIONS

This invention can be used to detect whether specific DNA sequences arepresent in the nucleic acid samples:

By heating the double-stranded sample nucleic acids to >94° C., the DNAmolecules will denature to single-stranded chains (or through othermeans to obtain single-stranded DNA). The resulting single-stranded DNAand reaction buffer are then flowed into the device described in thisinvention. Using temperature scanning methods, the melting temperaturesTm of every pair of the target and probes on the chip can be determined.If the target and probe are perfect match, the binding force between thetwo chains are maximum, resulting in the maximum Tm, which is equal tothe specific Tmp of the probe. If one (or more than two) mismatch existsbetween the target and the probe, the binding force will be less, andthe corresponding melting temperature Tm will be lower than the Tmp. Inother words, the standard to judge whether the result is reliable is tocompare the Tm with the specific Tmp of each probe. Therefore, thismethod will tell whether the corresponding target oligos to the probeson the chip exist in the sample nucleic acids.

This invention can also be used to separate different DNA molecules inthe sample:

Incubate single-stranded target DNA and the reaction buffer in thechamber described in this invention, and only select for the chipcontaining probes with a single Tmp. Temperature scanning is then usedto monitor the Tm of the target and probe oligos at various spots on thechip. Disregard the washing buffer when the Tm is less than Tmp.Therefore, the buffer eluted at or greater than the specific Tmp shouldcontain those target DNA molecules with perfect matches to the probes onthe chip robes on the chip.

1. Method for detecting DNA hybridization on DNA chips using4-Dimensional parameters which comprises the following steps: (1)placing a DNA chip containing probes in a temperature-controlledchamber; (2) incubating the DNA chip with single-stranded target oligosand a fluorescent dye in a reaction buffer, wherein the fluorescent dyeinserts into double-stranded DNA molecules; (3) lowering the temperaturein the temperature-controlled chamber to the annealing temperature (Th),wherein the single-stranded target oligos hybridize to the probes on thechip to form double-stranded DNA molecules and wherein the fluorescentdye inserts into the double-stranded DNA molecules thereby, labeling thedouble-stranded DNA molecules with the fluorescent dye; (4) increasingthe temperature in the temperature-controlled chamber from Th to 100° C.at the speed of 0.001–1° C./sec, (5) scanning the chip at each ΔT° C. bya scanner to obtain a fluorescent intensity F, wherein when thetemperature in the termparature-controlled chamber reaches the meltingtemperature Tm for a given probe, the double-stranded DNA moleculedissociates and the fluorescent dye diffuses into the buffer, resultingin a rapid loss of fluorescent intensity; wherein the scanning offluorescent intensity of every probe on the chip records the turningpoint on a continuous melting curve or a derivative melting curve foreach of the double-stranded DNA molecules, which provides the Tm of eachof the double-stranded DNA molecules at the peak position on the curves;and (6) comparing the Tm for each probe from step (5) with the knownoptimum melting temperature for each of probe that hybridzes perfectlywith a complementary target oligo (Tmp) to detect the presence of theperfectly complementary target oligo, wherein the target oligo ispresent when the Tm equals Tmp for a given probe.
 2. The method of claim1, which further comprises washing the DNA chip at the annealingtemperature to wash off the excess target oligos, fluorescent dye andreaction buffer.
 3. The method of claim 1, wherein the annealingtemperature Th is in the range of 4–89° C.
 4. The method of claim 1,wherein the melting temperature Tm is in the range of 8–100° C.
 5. Themethod of claim 1, wherein the temperature increasing speed is at0.01–1° C./sec.
 6. The method of claim 1, wherein the fluorescent dye isSYBR Green I, SYBR Green II, or SYBR Gold.
 7. A device adapted forperforming the method of claim 1 comprising a transparent glass box, atemperature controlled themocycler device connected to the glass box,and a buffer input and a buffer output connected to the glass box. 8.The device of claim 7, wherein the Temperature Controlled thermocyclerdevice comprises a Temperature Sensor, a a thermocycler and aTemperature Controlling Device; the temperature sensor and thetemperature controlling device are connected, and the temperaturecontrolling device and the thermocycler are also connected.
 9. Thedevice of claim 7 for analyzing and separating DNA samples.
 10. Thedevice of claim 7 for analyzing and separating RNA samples.
 11. Thedevice of claim 7 which further comprises DNA chips.